Beta-adrenergic receptor (AR) is a pivotal regulator of cardiac function. However, enhanced beta1-AR signaling promotes apoptotic heart cell death, which is implicated in myocardial remodeling and heart failure. The goal of this study is to determine whether the classic Gs-adenylyl cyclase-cAMP-PKA pathway is essential to beta1-AR apoptotic effect. To avoid complicated interactions between beta-AR subtypes, we created a genetically "pure" beta1-AR experimental setting by expressing the mouse beta1-AR in the null background of beta1/beta2-AR double knockout adult mouse cardiomyocytes using adenoviral gene transfer. Here we show that beta1-AR stimulation is markedly increases myocyte apoptosis, as evidenced by increased TUNEL staining positive cells, DNA fragmentation (cell death ELSIA and DNA laddering). Beat1-AR-induced apoptosis is abolished by a beta-AR antagonist, propranolol (1 uM). To our surprise, specific PKA inhibitors, including Rp-CPT-cAMP (100 uM), H89 (5 uM) and a peptide inhibitor (PKI, 5uM), while blocking forskolin (0.1 uM)-evoked myocyte apoptosis, cannot prevent beta1-AR-induced apoptotic cell death. Ironically, blocking Ca2+ influx by a L-type Ca2+ channel inhibitor nifidipine (1 uM) or buffering intracellular Ca2+ with EGTA-AM (1 uM) fully protects heart cells against beta1-AR-mediatd apoptosis, suggesting that a PKA-independent increase in intracellular Ca2+ is obligatory to beta1-AR apoptotic effect. To delineate the downstream events of beta1-AR/Ca2+ apoptotic signaling, we first evaluated the potential role of a Ca2+/ calmodulin-dependent phosphatase, calcineurin, since this phosphatase has been implicated in beta-AR-induced apoptosis in cardiomyocytes. However, inhibition of calcineurin with cyclosporin A (5 uM) or FK506 (10 uM) does not affect b1-AR-mediated apoptosis. In sharp contrast, inhibition of Ca2+/calmodulin kinase II (CaMKII) with KN-93 (0.5 uM) or a peptide inhibitor (AIP, 10uM) fully abolishes b1-AR-promoted apoptotic cell death. Further, beta1-AR induces a robust increase in CaMKII activity in a PKA-independent manner. Thus, beta1-AR apoptotic effect is mediated by a CaMKII-, rather then PKA-, dependent mechanism. These findings shed light on our understanding of beta1-AR cardiac detrimental effects, also underscore novel therapeutic strategies for the treatment of heart failure.